Int J Biol Med Res.

2023 ;14(2):7575- 7581

Int J Biol Med Res www.biomedscidirect.com
Volume 14, Issue 2, April 2023

Contents lists available at BioMedSciDirect Publications

International Journal of Biological & Medical Research

Journal homepage: www.biomedscidirect.com
BioMedSciDirect International Journal of
Publications BIOLOGICAL AND MEDICAL RESEARCH

Original article
Diagnostic tool development using advanced techniques in biotechnology for
microbial keratitis
V.Nithya,* Anusha Bhaskar
Assistant Professor in Microbiology, Urumu Dhanalakshmi College, Kattur, Tiruchirappalli, Tamil Nadu, India.

ARTICLE INFO ABSTRACT

Keywords:
Purpose: To study the demographic characteristics, associated factors, causative agents, of
Ocular infection
infectious keratitis and develop a diagnostic tool to aid easy diagnosis of keratitis.Methods:
keratitis
bacterial keratitis Corneal scrapes were collected and subjected to culture, microscopy, considering age,
mycotic diseases occupation, geographical, Frequency of predisposing ocular conditions, antibiotic
multiplex PCR susceptibility test, resistance patterns test, drug sensitivity and 16s r-DNAas well as 18s r-DNA
based identification was performed and submitted to data bank with accession number. The
16S rDNA sequences of the individual bacteria and fungi were used for a universal primer
design and thereby multiplex PCR can be performed.Results: A total of 250 consecutive patients
with infective keratitis were evaluated, of which 77 (30.8%) were found to be of bacterial, 67
(26.8%) were fungal, 16 (6.4%) were both fungal and bacterial, and the remaining 90 (36%)
were found to be culture negative. Contact lens wear was the main risk factor (80.8%). Ocular
surface disease (23.6%), ocular trauma (14.8%), corneal surgery (4.4%) and corneal suture
(6.4%) of cases were found in corneal ulcers. Most community acquired bacterial and fungal
ulcers resolve with appropriate treatment. 64% of the infections involved positive cultures and
36% involved negative cultures, were found in polymicrobial mode of infection. Fusarium spp.
(32.75%) was the most predominant species followed by Aspergillus sp. (20.68%) was found in
fungal corneal ulcers and also Staphylococcus sp. was the most common bacteria found in
bacterial cultures. The 16S rDNA sequence of the bacteria and fungi cultured from the isolates
of the corneal scrapping were performed and the genes were submitted in Genbank.The
primers designed for bacteria and fungi gave good results in the multiplex PCR carried out and
we suggest this can be used as a diagnostic tool for keratitis.

c
Copyright 2010 BioMedSciDirect Publications IJBMR - ISSN: 0976:6685. All rights reserved.

Introduction

Corneal infection is one of the leading cause of visual loss [1] an
estimated 6.8 million people suffer from keratitis in India and the
number is expected to increase up to 10.6 million by 2020 [2].
Bacterial keratitis is the most common form of suppurative
corneal ulceration and a variety of fungi are also implicated in this
infection. Infective keratitis especially of mycotic origin has been
reported from diverse climatic zones, it appears to be more
common in subtropical and tropical regions. A global increase in
the incidence of this disease has been noted in recent decades,
mainly due to the topical use of antibacterial and steroidal
preparations [3] which compromise the cornea to opportunistic
fungal infections.
* Corresponding Author : *Dr. V.Nithya
Assistant Professor in Microbiology, Urumu Dhanalakshmi College,
Kattur, Tiruchirappalli, Tamil Nadu, India.
E-mail: nithyavaradharaj20@gmail.com
c Copyright 2011. CurrentSciDirect Publications. IJBMR - All rights reserved.
While many organisms are capable of causing ocular infection
microbiologic examination of clinical specimens is required for
diagnosis. Microbiology tests are successful in identifying the
causative organism; however, Multiplex PCR has been shown to be
especially suited for detecting small amounts of microbial DNA
present in ocular specimens [4-10]. This is particularly true for
viruses that set up infection of the eye. While only a limited number
of viruses especially, cytomegalovirus, herpes simplex virus and
zoster virus which are the etiologic agents.
A large number of bacterial and fungal pathogens are
commonly encountered during diagnosis of keratitis. The 16S
subunit of rDNA of bacteria and 18S subunit (ITS regions) of the
fungi are the most conserved region that can different one species
from another [11]. Our study aims at using PCR amplification and
sequence analysis of 16S rDNA to detect bacterial and 18S rDNA to
detect fungal pathogens in subjects with keratitis. The amplified
sequences were deposited in the GenBank. A unique primer was
designed for each organism and efforts are underway to design a
universal primer for bacteria and fungi which will enable us to
 V.Nithya & Anusha Bhaskar et al./Int J Biol Med Res.14(2):7575- 7581
develop a diagnostic tool for easy diagnosis of the nature of
keratitis by multiplex PCR. This analysis will ascertain the
organism(s) responsible for the infection and also help in the
diagnosis of mixed infections.
MATERIALS AND METHODS
Study population and sample collection
In this study 250 consecutive outpatients 143 males and 107
females were with corneal ulcerations were examined in the Eye
Clinic of the General Hospital of Tiruchirappalli from January 2009
to February 2012. This study was approved by the Research Ethics
Committee, of the Hospital. An ophthalmic history, including risk
factors for corneal ulceration (ocular surface disease, previous
ocular surgery, contact lens use of trauma) and use of antibiotics
or steroids was obtained from each patient. All patients were
examined under a slit-lamp biomicroscope by an ophthalmologist.
Corneal scrapings were collected after instillation of 4%
lignocaine without preservative under aseptic conditions from
each ulcer by an ophthalmologist using a sterile Bard Parker blade
(No 15). Scrapings were performed under magnification of slit-
lamp or operating microscope. Leading edge and base of each
ulcer were scraped initially and the material obtained were
directly inoculated onto the surface of solid media such as sheep
blood agar, chocolate agar and Sabouraud Dextrose Agar (SDA) in
a row of C- shaped streaks and also deep inoculation in the liquid
media such as brain heart infusion (BHI) broth without
gentamycin sulphate and thioglycollate medium. Subsequent
scrapings were spread onto labeled slides in a thin, even manner
for 10 % potassium hydroxide (KOH) wet mount and Gram
staining. Control patients were defined as patients who had
epithelial defects of noninfectious etiologies or infective keratitis.
The control subjects were age matched.
Primary Screening
All bacterial cultures were incubated aerobically. Cultures on
chocolate agar were evaluated at 24 - 48 hours and then discarded
if no growth was seen. All media were incubated at 35°C (±1)
except SDA, which are incubated at 27°C (±1) in BOD incubator.
Cultures inoculated in BHI broth were examined for turbidity and
subsequently sub cultured and gram stained for identification.
Determination of positive culture samples were done according to
methods by Bharathi et. al [12].
Secondary Screening (Culture based)
The specific identification of bacterial pathogens was based on
microscopic morphology, staining characteristics, and
biochemical properties using standard laboratory criteria. Fungi
were identified by their colony characteristics on SDA and by their
microscopic appearance in Lacto phenol cotton blue.
Antibacterial sensitivity test
Antibiotic susceptibility testing of each isolate was done using
routine antibiotics discs Gentamycin; Ampicillin; Tetracycline; Co-
trimaxazole; Ceftriaxon; Cephalotin; Cefotaxime; Ceftriaxon;
Kanamycin according to disc diffusion technique by Kirby Bauer
method on Muller Hinton agar (MHA) and were evaluated by as
per the standards [13].
Anti-fungal drug sensitivity test
Various antimicrobials were incorporated in the study to
check the sensitivity of the filamentous isolates. The diffusion
method has been employed using tablets of amphotericin B,
7576
clotrimazole, econazole, flucytosine, miconazole, and nystatin.
These diffusion tablet tests were performed as recommended by
Casals [14]. For all cases, post diagnosis antimicrobial therapy was
given. Subsequent treatment was tailored according to the
microbiological diagnosis and sensitivity results. The final visual
acuity was defined as the visual acuity on discharge from the ward
(Not documented in this work).
Molecular Identification
In this study aliquots from positive cultures were obtained
from the keratitis.. For most sequencing experiments a 'universal'
primer was used, this being one that was complementary to the
part of the vector DNA immediately.
In most cases, agarose gel electrophoresis is used for the
separation of DNA fragments ranging from 10 kb~0.2 kb, a vertical
polyacrylamide gel is more appropriate henece used. The
amplification of 16S r-DNA of any bacterial species is possible
without prior cultivation when broad-range PCR primers targeted
to highly conserved regions are applied[15]. Identified bacteria in
corneal scrapings and in vitreous samples of patients who had
keratitis and endophthalmitis by amplification and subsequent
direct sequencing of 16S r-DNA were performed by designing
specific primers for designated bacterial and fungal isolates.
Primer design for multiplex PCR
The primer for individual bacteria and fungi were designed
using Primer 3 software. Then using these individual primers a
common primer for bacteria and fungi were developed.
Reaction volume
Take five 0.2ml or 0.5 ml PCR tubes and added 0.5 μl of each
DNA sample, Distilled water 18.3µl, Taq DNA polymerase buffer
(10x) 2.5µl, dNTP mix (2mM) 2.5µl, 2 Universal Primers (10µM)
0.5µl for each, DNA polymerase enzyme (5U/µl) 0.2µl, Total
Reaction Volume 25.0µl to the respective PCR tube
Multiplex PCR
Multiplex PCR amplification was performed in a Gene Cycle
under the following conditions: first heat initial denaturation
94ºC for 5 minutes, cycle denaturation 94ºC for 45 seconds for 34
cycles, annealing 50ºC for 45 seconds and extension 72ºC for 1
minute. This was followed by incubation at 72 ºC for 10 minutes
and cooling at 4 ºC. Negative control reaction mixtures contained
sterile distilled water in place of template DNA. Finally resolve the
PCR product onto 1% agarose gel electrophoresis was used to
visualize multiplex PCR DNA product and photographed under UV
(Progen Biotech, Salem, Tamil Nadu). The universal primers were
synthesized at Sigma, Bangalore.
Results
Epidemiological characteristics
Out of 250 patients 143 (52.7 %) were males and 107 (42.8 %)
were females. There were 153 (61.5%) rural residents and
97(38.5%) urban residents. Patients above the age of 50 years 115
(46 %) were significantly less than patients below 50 years 135
(54 %) were shown in Figure 1. Non-agricultural workers (Figure
2) were significantly less in number than were farmers 108 (54%).
Frequency of predisposing ocular conditions in bacterial
keratitis
Contact lens (CL) wear was the most common risk factor. This
was encountered in 127 eyes (50.8%). Soft CL was noted in 41.7%
 V.Nithya & Anusha Bhaskar et al./Int J Biol Med Res.14(2):7575- 7581
7577

of cases, extended wear contact lens in 44.8%, and disposable Antifungal sensitivity test
contact lens wear in 13.5% of cases were found in CL cases (Table
Antifungal sensitivity test as were performed and Fusarium
1).
spp. were found to be more resistance, while Candida spp. is most
In 59 cases of ocular surface diseases, 45.8% cases of sensitive among all isolates. In these tests the Azoles drugs were
aphakic bullous keratopathy, 16.9% cases of epidermal dysplasia most sensitivity organisms when compared with Polyenes
and 37.3% cases of chronic bullous keratopathy were found. In 37
Molecular identification
cases of ocular trauma, 59.1% of workplace-related trauma,
13.9% of home-related trauma and 27% motor vehicle-related In order to develop the proposed methodology, the primers for
trauma were found in corneal patients. In 11 cases of corneal conserved regions of the rDNA for the bacterial and fungal
related surgery, 45.5% of Penetrating Keratoplasty (PK), 36.4% of pathogens were synthesized. The DNA isolated from the bacteria
Anterior Lamellar Keratoplasty (LK) and 18.1% Endothelial and fungi were amplified and sequenced and the sequences were
Keratoplasty (EK) were found. In 16 cases of corneal sutures, used for the strain identification. A BLAST was carried out to
18.8% of facial nerve plasy (leprosy), 31.2% of eyelid trauma, confirm the organism from the database. The sequences were
12.5% of Exposure keratopathy, 12.5% of Secondary to cataract submitted to the GenBank. A unique primer was designed for
extraction and 25% of Thyroid Exophthalmopathy were found in bacteria and fungi and multiplex PCR was carried out and the
corneal ulcers are shown in Table 2. results obtained are shown in Fig 3. This could be positively
developed into an unique diagnostic tool for identification of the
Microbiological diagnosis
causative organism of keratitis.
Cultures were positive and fulfilled the criteria established for
the presence of infection in 110 (55%) of the 200 corneal ulcers Table 1: Frequency of predisposing ocular conditions in
(Table 3). Pure bacterial growth was present in 70 (28%) of the bacterial keratitis
250 cultures performed and pure fungal growth in 67 (26.8%).
Mixed microbial growth was present in the cultures of 16 (6.4%)
of the 250 patients. A total of 354 bacterial organisms were
cultured from160 corneal ulcers (Table 4). Of the 70 isolates, 228
(64.5%) were Gram positive and 126 (35.5%) were Gram-
negative bacteria. Staphylococcus sp. was the most commonly
isolated bacterial organism representing 99 (66.3%) of all positive
bacterial cultures. The next most commonly isolated Gram-
positive organism was propionibacterium acnes with 31
(39.24%) positive cultures. Of these 31 cultures, 19 were pure, and
12 were mixed with fungi. Pseudomonas aeruginosa was isolated
from 40 cultures (34.48%) and followed by serratia sp. 38 cultures
Table 2: Ocular Factors Predisposing to Culture-Proven
were the most frequently occurring Gram-negative organism. A
Bacterial Keratitis
total of 116 fungal organisms were cultured from equal number of
corneal ulcers (Table 6) of which 38 (32.75%) were Fusarium sp.,
24 (20.68%) were Aspergillus sp., 17 (14.65%) were Candida sp.,
9 (7.75%) were Byssochlamys nivea, 10 (8.62%) were Sebipora
aquosa and 18 (5.51%) were A. fumigates were found in fungal
species. Of the 116 fungal ulcers 39 were positive for fungal
elements on KOH examination.
In CL wearers group, 65.3% of the corneal scrapings were
positive mostly Staphylococcus sp. (34.7) of isolated bacteria and
Gram negative, mostly Pseudomonas aeruginosa. Contact lens
and/or storage cases cultures were performed in 127 cases. In
ocular disease group, 67% of the corneal scrapings were positive
whereas 37% of isolated bacteria were found to be Gram negative.
In ocular trauma group, 58.2% of the corneal scrapings were
positive whereas 41.8% of isolated bacteria were Gram negative
was found. In corneal surgery group, 65.3% of the corneal
scrapings were positive whereas 34.7% of isolated bacteria were
Gram negative was found. In corneal sutures disease group, 72.7%
of the corneal scrapings were
Antibiotic Resistance in causative Bacteria
In vitro resistance to at least one antibiotic was found in 55
(33%) bacterial isolates (Table 8). Resistance to multiple
antibiotics was found in 250 isolates, S. aureus had the highest rate
of resistance, whereas no isolates of P. aeruginosa were resistant
to tested antibiotics. Scrapings from cases related to prior ocular
surgery were more likely to culture resistant bacteria than
scrapings related to other keratitis risk factors.
 V.Nithya & Anusha Bhaskar et al./Int J Biol Med Res.14(2):7575- 7581
7578

Table 5: Organisms isolated in bacterial corneal ulcers.

Polybacterial infection was noted in this cases

Table 3: Microbial growth pattern of cultures from corneal

ulcers (n=250) from the Eye clinic of the General Hospital in
Thiruchirappalli, Tamil Nadu, India.

Table 4: Bacterial isolates from corneal ulcers

 V.Nithya & Anusha Bhaskar et al./Int J Biol Med Res.14(2):7575- 7581
7579

Table 10: Drug sensitivity of fungi isolated from cases of

keratitis

GM: Gentamycin; AM: Ampicillin; TE: Tetracycline; SXT: Co-

trimaxazole; CRO: Ceftriaxon; CF: Cephalotin; CTX:
Cefotaxime; TOB: Ceftriaxon; K: Kanamycin; –: not used
 V.Nithya & Anusha Bhaskar et al./Int J Biol Med Res.14(2):7575- 7581
Fig 1 Age of patients with corneal ulceration (n=250) in the
Eye clinic of the General Hospital in Thiruchirappalli, Tamil
Nadu, India.
Fig 2 Occupation of patients with corneal ulceration (n=250)
in the Eye clinic of the General Hospital in Thiruchirappalli,
Tamil Nadu, India.
* An individual either male or female, who does heavy manual
labor, lifting loading & carrying of material usually balanced
on head;
† Middle class workers such as mechanics, stonemasons,
electricians, carpenters, pipe fitters, welders, and fishermen.
This category also includes profession as teachers, police,
office workers, factory workers, drivers and merchants
Fig:3 Electrophoresis of multiplex PCR products from
organisms causing keratitis
L1- DNA 100 bp step ladder (Promega)
Agarose Gel (1%) M – 1Kb DNA Ladder,AG – Aspergillus
fumigatus,CA – Candida albicans
FM – Fusarium sp., KA – Klebsiella pneumoniae, PM –
Pseudomonas aeruginosa
SA – Staphylococcus aureus,MPL – Multiplex PCR (for all
organisms
7580
DISCUSSION
This study evaluated demographic, clinical, microbiological,
and treatment information taken from medical records of all
patients that had corneal scrapings over a 3 year period at a public
hospital in Tiruchirappalli India. In this population, we found
there average age of patients was 51 years, and 60% of patients
were men. Contact lens wear and ocular trauma were more
common risk factors in young patients, whereas prior ocular
surface disease and previous ocular surgery were more common
risk factors in older patients.
This predominance of men presenting with traumatic keratitis
is reflected in another recent study, which found that 90% of
traumatic keratitis occurred in men.
There has been in the past several years a steady increase in CL
wearers. Subsequently, CL wear is now the major predisposing
factor for corneal infection in the United States and Western
Europe [16] and a matter of public health concern. Soft CL have
greatly increased the risk of bacterial keratitis, which is estimated
to be 10–20 times higher with the use of extended wear disposable
CL.8 hypoxia and hypercapnia of the cornea induced effects were
already reported in case of contact lense wearers [17].
Multiple organisms have been reported from the microbial
keratitis seen in association with CL wear. We found a moderate
shift towards higher prevalence of Gram negative rods compared
with that prevalence in the absence of CL wear and we confirmed
the results of previous studies. These results confirm that Gram
negative bacteria are more often associated with soft CL wear.
However, conversely to what might be expected, the percentage of
Gram negative organisms is lower than those of Gram positive
organisms. Approximately two thirds of CL related bacterial
keratitis are associated with Gram positive cocci, such as
staphylococci and streptococci and one third is associated with
Gram negative rods, especially Pseudomonas aeruginosa and
Serratia marcescens,
This result suggests that contact lenses and cases may tend to
preferentially harbour Gram negative organisms, but that the
causative organism may well not match that organism, and indeed
Gram positive organisms are important in contact lens related
keratitis.
Of 57 fungal isolates cultured from equal number of corneal
ulcers 29.82% were Fusarium spp., 21.05% were Aspergillus spp.
and there were no onfilamentous fungus because these are
specifically overcomes the Fusarium sp, is similar reported from
South Florida and from Ghana by previous researchers. The
numerous species occurred in india are like Fusarium spp. as the
predominant fungal species are reported by Bharathi et al [12].
More than three quarters of cases were treated initially on an
outpatient basis. All the patients have been traeted with good
traditional antibiotics. Thus most have undergoes the
combination of antibiotics, whereas broad spectrum antibiotic
therapy. There was no evidence of proven fungal keratitis above
the age of 61 yr. in north India reported in 2005 [18], but in this
study we have a broad spectrum of infection up to the age of 71 yrs
[19]. Status of antibiotic resistance also a huge interruption in the
line of treatment of keratitis with some routine antibiotics like
cyclohexamide, amphoterin, in case of fungal spectrum [20,21].
This case audit has defined the common risk factors, causative
organisms, antibiotic resistance, patient demographics, clinical
presentations, of patients with keratitis presenting to a major
hospital in region of south India.e as a best mode of diagnosis.
 V.Nithya & Anusha Bhaskar et al./Int J Biol Med Res.14(2):7575- 7581
7581

We, also investigated the possibility of using PCR amplification 15. Joseph J, Sharma S, Somasheila IM, Krishna PV, Garg P, Nutheti R, Kenneth J,
Balasubramanian D. Microsporidial keratitis in India: 16S rRNA gene-based
and 16S rDNA and 18S rDNA of bacteria and fungi respectively to PCR assay for diagnosis and species identification of microspordia in clinical
identify the microorganisms in the setting of the disease. The PCR samples. Invest Ophthal and Visual Science 2006; 47: 4468 – 4473.
based technique has a certain advantage over the standard culture 16. Jones DB. Pathogenesis of bacterial and fungal keratitis. Trans Ophthalmol
methods because the results are obtained with shorter time than Soc UK 1978; 98: 367-71.
the conventional methods. We have sequenced the conserved 17. Wood TO, Williford W. Treatment of keratitis with amphotericin B 0.15%.
regions of the rDNA and they have been submitted to the Genbank. Am J Ophthalmol 1976; 75: 847-49
On the basis of the results obtained we have designed a primer one 18. Chowdhary A , Singh K. Spectrum of Fungal Keratitis in North India, Cornea:
common for bacteria and one for fungi and the multiplex PCR 2005 – 24; 8-15
carried out gave very good and reproducible results. 19. Kaufman E, Wood RM. Mycotic keratitis. Am J Ophthalmol 1965; 59: 993-
1000.
Conclusion
20. Flynn JR. Fusarium keratitis treated with cycloheximide. Am J Ophtbalmol
The clinical histories, an agricultural status for most patients, 1964; 58: 637-41.
recollection of a preceding trauma from vegetable matter and soil 21. Iwata K. Drug resistance in human pathogenic fungi. Eur J Epidemiol 1992;
and the patterns of causative fungi and bacteria, viz., P. aeruginosa, 8: 407-21
S. penumoniae, among the bacteria and Aspergillus sp. Fusarium
oxysporum Penicillium sp. Paecilomyces farinosus among the
fungi. The filamentous fungi showed sensitivity in decreasing
order to flucytosine, nystatin, amphotericin B, and econazole. In
conclusion, this study demonstrated that culture-proven bacterial
keratitis mainly affected middle-aged males who sustained ocular
trauma. Gram-negative bacteria in general and Pseudomonas
aeruginosa in particular were the most common microorganisms
isolated. In this study we have carried out the molecular
identification of the organisms causing keratitis and we also
recommend multiplex PCR techniqu

References
1. Wilhelmus KR. Bacterial keratitis, p. 970–1031. In JS. Pepose, GN. Holland,
and KR. Wilhelmus editors. Ocular infection and immunity. Mosby Year Book
Inc., St. Louis, Mo. 1996
2. Whitcher JP, Srinivasan M, Upadhyay MP. Corneal blindness A Global
perspective. Bull WHO;2001;79;214-21
3. Chin GN, Hundiuk RA, Kwasny GP, Schultz GP. Keratomycosis in Wisconsin.
Am J Ophthalmol 1975; 79: 121-25.
4. Cunningham, ET, Short GA, Irvine AR. AIDS-associated herpes simplex virus
retinitis: clinical description and use of a polymerase chain reaction-based
assay as a diagnostic tool. Arch. Ophthalmol. 1996; 114:834–840.
5. Dean D, Millman K. Molecular and mutation trends analysis ofomp1 alleles
for serovar E of Chlamydia trachomatis. J. Clin. Invest. 1997; 99:475–483.
6. Dean D, Schachter JS, Dawson CR, Stephens RS.. Comparison of the major
outer membrane protein variant sequence regions of B/Ba isolates: a
molecular epidemiological approach to Chlamydia trachomatis infections. J.
Infect. Dis. 1992; 166:383–392.
7. Hykin PG, Tobal K, McIntyre G, Matheson MM, Towler HMA, Lightman SL.
The diagnosis of delayed post-operative endophthalmitis by polymerase
chain reaction of bacterial DNA in vitreous samples. J. Med. Microbiol. 1994;
40:408–415.
8. Knox CM, Margolis TP, Chandler D, Short GA. PCR based assays for the
diagnosis of viral retinitis: use in diagnostic dilemmas. Ophthalmology
1998;105:37–45.
9. McCann JD, Margolis TP, Wong MG. A sensitive and specific polymerase chain
reaction-based assay for the diagnosis of cytomegalovirus retinitis. Am. J.
Ophthalmol. 1995; 120:219–226.
10. Short GA, Margolis TP, Kuppermann BD. A polymerase chain reaction-based
assay for the diagnosis of varicella-zoster virus retinitis in patients with
AIDS. Am. J. Ophthalmol. 1997;123:157–164.
11. Relman DA. The identification of uncultured microbial pathogens.
J. Infect. Dis. 1993; 168:1–8.
12. Bharathi MJ, Ramakrishnan R, Vasu S; Meenakshi, Palaniappan R.
Aetiological diagnosis of microbial keratitis in South India - a study of 1618
cases. Indian J Med Microbiol.2002; 20(1):19-24.
13. Duru ME, Cakir A, Kordali S, Zengin H, Harmandar M, Izumi S, Hirata T.
Chemical composition and antifungal properties of essential oils of three
Pistacia species. Fitoterapia 2003; 74, 170 – 176.
14. Casal JB. Tablet sensitivity testing on pathogenic fungi. J Clin Pathol, 1979;
32,719 – 722. c Copyright 2019 BioMedSciDirect Publications IJBMR - ISSN: 0976:6685.
All rights reserved.